【摘要】：為探討組蛋白修飾對(duì)絲狀真菌里氏木霉(Trichoderma reesei,T.reesei)表達(dá)纖維素酶的表觀遺傳調(diào)控機(jī)制,本文通過(guò)基因敲除方法使組蛋白H3賴氨酸甲基轉(zhuǎn)移酶(histone H3 lysine methyltransferase,HKMT)和組蛋白去乙�；�(histone deacetylase,HDAC)的基因缺失,成功篩選獲得突變菌株Δhkmt與Δhdac;通過(guò)siRNA技術(shù)干擾組蛋白乙�；�(histone acetylase,HAT)的基因gcn5并成功篩選出一株干擾效果較好的菌株T.reesei-gcn5-T10。通過(guò)顯微鏡觀察突變菌株Δhkmt與Δhdac以及干擾重組菌株T.reesei-gcn5-T10的菌絲的變化;通過(guò)濾紙酶活(Filter paper enzymatic activities,FPA)和羧甲基纖維素鈉酶活(Carboxymethyl Cellulose-Na enzymatic activities,CMCA)檢測(cè)纖維素酶的活性變化;實(shí)時(shí)熒光定量PCR(Real-time fluorescent quantitative PCR,RT-qPCR)檢測(cè)了突變菌株Δhkmt與Δhdac以及干擾重組菌株T.reesei-gcn5-T10中纖維素酶基因cbh1、egl1與酶激活因子xyr1的mRNA表達(dá)水平,以及hkmt、hdac與gcn5的mRNA水平的變化,探討他們與纖維素酶表達(dá)之間的關(guān)系。結(jié)果顯示,(1)在相同倍數(shù)的物鏡下觀察到突變菌株Δhkmt與Δhdac的菌絲均長(zhǎng)于出發(fā)菌株T.reesei QM9414,并且分支較多而長(zhǎng)。而干擾重組菌株T.reesei-gcn5-T10的菌絲相較于出發(fā)菌株T.reesei QM9414,變短而粗,并且末端膨大,內(nèi)部變渾濁。(2)突變菌株Δhkmt與Δhdac的FPA和CMCA明顯高于出發(fā)菌株T.reesei QM9414。突變菌株Δhkmt各時(shí)段比出發(fā)菌株T.reesei QM9414分別平均高出5.00 IU/m L和15.00 IU/mL;突變菌株Δhdac各時(shí)段比出發(fā)菌株T.reesei QM9414分別平均高出6.50 IU/m L和15.00IU/mL。而干擾重組菌株T.reesei-gcn5-T10的FPA和CMCA明顯低于出發(fā)菌株T.reesei QM9414,分別平均低出10.00 IU/mL和5.20 IU/mL。(3)RT-qPCR結(jié)果顯示,突變菌株Δhkmt中纖維素酶基因cbh1、egl1與酶激活因子xyr1基因的相對(duì)表達(dá)量均高于出發(fā)菌株T.reesei QM9414,分別高出4.51倍、3.87倍和2.51倍,突變菌株Δhdac中cbh1、egl1與xyr1基因表達(dá)量也均高出發(fā)菌株T.reesei QM9414,分別高出6.50倍、6.01倍和4.51倍,而干擾重組菌株T.reesei-gcn5-T10中cbh1、egl1與xyr1基因表達(dá)量均低于出發(fā)菌株T.reesei QM9414,分別為原菌的0.34倍、0.84倍以及0.44倍。(4)RT-qPCR對(duì)hkmt、hdac與gcn5的分析結(jié)果顯示,干擾gcn5,即HAT表達(dá)量下降,導(dǎo)致hdac表達(dá)量上升,hkmt的表達(dá)量也隨之升高。結(jié)論:hkmt基因與hdac基因被敲除后,組蛋白的甲基化修飾與去乙�；揎棻灰种�,纖維素酶基因cbh1,egl1與激活因子xyr1基因的表達(dá)水平顯著增加,最終激活纖維素酶的表達(dá)。而gcn5被干擾后,組蛋白乙酰化修飾被抑制,使得相關(guān)基因表達(dá)顯著下降,從而導(dǎo)致纖維素酶活下降。
[Abstract]:To investigate the epigenetic regulation mechanism of histone modification on the expression of cellulase in filamentous fungus Trichoderma reei. The gene deletion of histone H3 lysine methyltransferase and histone deacetyltransferase (HDAC) was studied by gene knockout. The mutant strain 螖 hkmt and 螖 hdac were successfully screened, and a strain T.reesei-gcn5-T10 was successfully screened by siRNA technique which interfered with the gene gcn5 of histone acetylase. The changes of 螖 hkmt and 螖 hdac and mycelium interfering with recombinant strain T.reesei-gcn5-T10 were observed by microscope, and cellulase activity was detected by (Filter paper enzymatic actives and Carboxymethyl Cellulose-Na enzymatic actives. Real time fluorescence quantitative PCR (PCR) was used to detect the mRNA expression levels of cellulase gene cbh1negl1 and activator xyr1 in mutant strains 螖 hkmt and 螖 hdac, and the levels of mRNA of hkmttHDAC and gcn5 in T.reesei-gcn5-T10. To explore the relationship between them and cellulase expression. The results showed that (1) the hypha of 螖 hkmt and 螖 hdac were longer than that of the original strain T.reesei QM9414 under the same objective lens, and the branches were more and longer than those of the original strain T.reesei QM9414. Compared with the original strain T.reesei QM9414, the mycelium that interfered with the recombinant strain T.reesei-gcn5-T10 became shorter and thicker, and the ends expanded and the interior became turbid. (2) the FPA and CMCA of the mutant 螖 hkmt and 螖 hdac were significantly higher than those of the original strain T.reesei QM9414. The 螖 hkmt of mutant strain was 5.00 IU/m L and 15.00 IU/m / mL higher than that of the original strain T.reesei QM9414, and the 螖 hdac of the mutant was 6.50 IU/m L and 15.00 IU / mL higher than that of the original strain T.reesei QM9414 in each stage, respectively. The FPA and CMCA of the interfering recombinant strain T.reesei-gcn5-T10 were significantly lower than that of the original strain T.reesei QM9414, which were 10.00 IU/mL and 5.20 IU / mL 路(3) RT-qPCR, respectively. The relative expression of cellulase gene cbh1negl1 and enzyme activator xyr1 in 螖 hkmt was higher than that of original strain T.reesei QM9414, 4.51 times higher than that of T.reesei QM9414, 3.87 times and 2.51 times higher than that of original strain T.reesei QM9414, respectively. The expression of cbh1negl1 and xyr1 genes in 螖 hdac was also higher than that of T.reesei QM9414, which was 6.50 times higher than that of T.reesei QM9414 and 4.51 times higher than that of T.reesei QM9414, respectively. However, the expression of cbh1egl1 and xyr1 genes in T.reesei-gcn5-T10 was lower than that of the original strain T.reesei QM9414, which was 0.34 times and 0.44 times of the original strain, respectively. (4) the results of RT-qPCR analysis of hkmttn5 and gcn5 showed that the expression of HAT decreased. As a result, the expression of hdac increased and the expression of hkmt increased. Conclusion the methylation modification and deacetylation modification of histone were inhibited after the knockout of the two genes, and the expression level of cellulase gene cbh1egl1 and activator xyr1 increased significantly, and finally the expression of cellulase was activated. After gcn5 was interfered, histone acetylation modification was inhibited, and the expression of related genes was significantly decreased, which resulted in the decrease of cellulase activity.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q55

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 史晨霞;薛濤濤;趙秀娟;周媛;梁棟;崔向軍;;釀酒酵母組蛋白H3K14乙�；瘜�(duì)H3K4三甲基化的影響[J];信陽(yáng)師范學(xué)院學(xué)報(bào)(自然科學(xué)版);2016年02期

2 李瑋婷;張才成;李俊明;;小干擾RNA靶向系統(tǒng)的研究進(jìn)展[J];廣東醫(yī)學(xué);2013年17期

，

本文編號(hào)：2197742